Lactoferrin and Its Detection Methods: A Review.

Lactoferrin (LF) is one of the major functional proteins in maintaining human health due to its antioxidant, antibacterial, antiviral, and anti-inflammatory activities. Abnormal levels of LF in the human body are related to some serious diseases, such as inflammatory bowel disease, Alzheimer's disease and dry eye disease. Recent studies indicate that LF can be used as a biomarker for diagnosis of these diseases. Many methods have been developed to detect the level of LF. In this review, the biofunctions of LF and its potential to work as a biomarker are introduced. In addition, the current methods of detecting lactoferrin have been presented and discussed. We hope that this review will inspire efforts in the development of new sensing systems for LF detection.

Lactoferrin in cerebrospinal fluid and saliva is not a diagnostic biomarker for Alzheimer's disease in a mixed memory clinic population.

BACKGROUND: The pathological changes in Alzheimer's Disease (AD) and other neurodegenerative disorders begin decades prior to their clinical expression. However, the clinical diagnosis of neurodegenerative dementias is not straightforward. Lactoferrin is an iron-binding, antimicrobial glycoprotein with a plethora of functions, including acting as an important immune modulator and by having a bacteriocidic effect. Two previous studies indicated that salivary lactoferrin could differentiate between neurodegenerative dementias. METHODS: A total of 222 cerebrospinal fluid (CSF) and saliva samples from a consecutive, mixed memory clinic population were analysed for lactoferrin. In addition, the association between lactoferrin in CSF and saliva and the concentration of tau, phosphorylated tau (p-tau) and amyloid 1-42 (Aß42) in CSF were addressed. FINDINGS: CSF lactoferrin was assessed for the first time in a cohort of patients with neurodegenerative dementias. No significant differences were found in the levels of CSF or saliva lactoferrin between the diagnostic groups. In addition, no significant relationships were found between lactoferrin levels and tau, p-tau and Aß42, respectively. INTERPRETATION: Neither CSF nor saliva lactoferrin could differentiate between neurodegenerative dementias in this study.

Calprotectin and lactoferrin in the cerebrospinal fluid; biomarkers utilisable for differential diagnostics of bacterial and aseptic meningitis?

BACKGROUND: The aim of our work was to assess the diagnostic contribution of calprotectin and lactoferrin determinations in the cerebrospinal fluid when distinguishing between bacterial and aseptic meningitides. METHODS: In 23 patients with bacterial meningitis (BM) and in 50 patients with aseptic meningitis (AM), we determined the concentrations of calprotectin, lactoferrin and the conventional biomarkers like glucose, total protein, lactate and polynuclear count in the cerebrospinal fluid (CSF). The discriminative power of the various parameters studied was determined by means of receiver operating characteristic (ROC) curves: the area under the curve (AUC), sensitivity, specificity, the positive likelihood ratio (+LR), and the negative likelihood ratio (-LR). RESULTS: The diagnostic efficiency of calprotectin, lactoferrin, lactate, and polynuclear count when distinguishing between bacterial and aseptic meningitides, expressed by ROC curve parameters, was as follows: AUC (0.736, 0.946, 0.932, 0.932), sensitivity (86.2, 96.6, 90.0, 89.7), specificity (58.5, 92.4, 87.0, 90.6), +LR (2.08, 12.8, 6.9, 9.50), -LR (0.24, 0.04, 0.11, 0.11), respectively. The optimal cut point for calprotectin and lactoferrin was 191 ng/mL and 17.8 ng/mL, respectively. CONCLUSIONS: Our findings show, that the determination of lactoferrin in the CSF was diagnostically the most efficient marker in distinguishing between bacterial and viral meningitides. Calprotectin was far less efficient diagnostic marker. The polynuclear count and lactate concentration showed a very good diagnostic efficiency as well. The determination of protein and glucose was diagnostically less beneficial.

Sleep disorders in Parkinson's disease: clinical features, iron metabolism and related mechanism.

OBJECTIVE: To investigate clinical features, iron metabolism and neuroinflammation in Parkinson's disease (PD) patients with sleep disorders (SD). METHODS: 211 PD patients were evaluated by Pittsburgh Sleep Quality Index (PSQI) and a body of scales for motor symptoms and non-motor symptoms. 94 blood and 38 cerebral spinal fluid (CSF) samples were collected and iron and its metabolism-relating proteins, neuroinflammatory factors were detected and analyzed. RESULTS: 136 cases (64.5%) of PD patients were accompanied by SD. Factor with the highest score in PSQI was daytime dysfunction. Depression, restless leg syndrome, autonomic symptoms and fatigue contributed 68.6% of the variance of PSQI score. Transferrin level in serum and tumor necrosis factor-α level in CSF decreased, and the levels of iron, transferrin, lactoferrin and prostaglandin E2 in CSF increased in PD patients with SD compared with those without SD. In CSF, prostaglandin E2 level was positively correlated with the levels of transferrin and lactoferrin, and tumor necrosis factor-α level was negatively correlated with the levels of iron, transferrin and lactoferrin in CSF. CONCLUSIONS: Depression, restless leg syndrome, autonomic disorders and fatigue are the important contributors for the poor sleep in PD patients. Abnormal iron metabolism may cause excessive iron deposition in brain and be related to SD in PD patients through dual potential mechanisms, including neuroinflammation by activating microglia and neurotoxicity by targeting neurons. Hence, inhibition of iron deposition-related neuroinflammation and neurotoxicity may cast a new light for drug development for SD in PD patients.

Trans-endothelial and trans-epithelial transfer of lactoferrin into the brain through BBB and BCSFB in adult rats.

The transportation of intravenously administered bovine lactoferrin (bLF) into the cerebrospinal fluid (CSF) was immunohistochemically investigated in adult rats. Administered bLF was detected in the vesicular membranes of endothelial cells in cerebral blood vessels 10 min after the infusion. Numerous immunoreactive small vesicles were also detected at the ependymal cells in the choroid plexus. Moreover, the bLF concentration in the CSF was significantly increased at 1-2 hr after the intravenous infusion of bLF (10 or 30 mg/kg). These findings clearly demonstrate that LF is possibly transported into the brain matter even in adult animals.

[Level of lactoferrin in serum and cerebrospinal fluid of patients with meningitis].

Study showed 9-fold increase of concentration of lactoferrin (LF) in serum of patients with bacterial meningitis (BM) compared with normal concentration and 5-fold increase of LF concentration in patients with aseptic meningitis (AM). Level of LF in cerebrospinal fluid (CSF) of patients with BM and AM was 200-fold and 22-fold higher than in control group respectively. In 71% of patients with AM concentration of protein in CSF did not exceed minimal level observed in patients with BM. Level of LF in serum and CSF during treatment statistically significantly decreased. Concentration of LF (the latter is marker of neutrophilic granulocytes activation) can be used as a characteristic of acuteness and intensity of inflammatory process in central nervous system, whereas detection of LF in CSF--as additional criterion in differential diagnostics between bacterial and viral meningitis. Furthermore, repeated measurement of LF level can be useful for monitoring of disease course and assessment of effect of treatment.

Receptor-mediated transport of lactoferrin into the cerebrospinal fluid via plasma in young calves.

Milk, especially colostrum, contains different kinds of macromolecules abundantly, such as immunoglobulin G (IgG), lactoferrin (Lf), transferrin (Tf), and growth factors. These are essential for the development and maintenance of health, which greatly depends on the absorption and transportation of macromolecules to the target organs. To evaluate the macromolecular transport, and concentrations in plasma and cerebrospinal fluid (CSF), colostrum was fed to newborn calves followed by milk and milk replacer, and maintained up to the 4th week under farm conditions. Plasma and CSF were collected at different times, and were analyzed for Lf, Tf, IgG and iron concentrations. Lf, Tf and IgG concentrations were steeply increased in plasma and CSF after colostrum feeding, and fluctuating patterns were observed during the experiments. Furthermore, intraduodenal administration of bovine Lf alone in young calf experiments revealed that the Lf concentration reached a peak at 4 hr, and was 7 and 4 times higher than preadministration in plasma and CSF, respectively. To explore the transport mechanism of Lf into CSF in young calves, epithelial membranes of the choroid plexus were prepared and a binding assay for Lf receptors (Lf-R) was carried out with 125I-Lf. The saturation kinetics revealed that the Bmax of epithelial membranes was 26.15 nmol/mg protein with a Kd of 0.11 microM, which also showed that Lf-R is saturable and specific. Scatchard plot transformation showed the presence of a single type of Lf-R in the choroid plexus. These results suggest that Lf is transported into the CSF through receptor mediated transcytosis in young calves, and that Lf may play an important role(s) in brain function.

[Lactoferrin and its role in the pathogenesis of tick-borne encephalitis]. / Laktoferrin i ego diagnosticheskaia rol' pri kleshchevom éntsefalite.

The content of lactoferrin (LF) was studied in the liquor and blood of patients with tick-borne encephalitis (TBE) of the meningeal and focal types; additionally, the information density of the discussed parameter was assessed for evaluating the severity and degree of the inflammation process in the central nervous system (CNS). The LF level was determined in liquor of 37 samples obtained from TBE patients (main group) and of 10 persons with osteochondrosis (controls); it was also determined in the serum taken from 21 TBE patients and from 40 healthy donors by using the immune-enzyme analysis. The LF concentration in TBE patients was found to exceed the normal value by 1.5-3 times during the whole observation period. As for the liquor, it was high, by the onset of the disease, by more than 20 times, however, after the 7th day it was higher 6-fold. A direct dependence of a concentration of the studied protein on a form and severity of the disease was established. The LF level in the liquor of TBE patients alongside with clinical signs can be an objective indicator of a severity and activity of the inflammation process in the CNS; it can also be a criteria of how much the conducted therapy effective is.

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs.

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.

Elevated CSF lactoferrin in superficial siderosis of the central nervous system.

This study sought to identify abnormalities in the levels of iron transport proteins in patients with superficial siderosis of the central nervous system. We compared patients with superficial siderosis (n = 7) with patients suffering from various other neurological disorders (n = 176, total). CSF and serum levels of lactoferrin, and CSF levels of transferrin were measured by enzyme-linked immunosorbent assay. Serum transferrin was measured by nephelometry. Lactoferrin, but not transferrin, levels in the CSF were significantly elevated in superficial siderosis. Unexpectedly, CSF transferrin was decreased in multiple sclerosis patients. Enhanced CSF lactoferrin may reflect an increased iron transport requirement in the central nervous system in superficial siderosis and might be a useful measure for monitoring response to therapy.

Levels of antimicrobial molecules defensin and lactoferrin are elevated in the cerebrospinal fluid of children with meningitis.

OBJECTIVE: To measure levels of defensins and lactoferrin in the cerebrospinal fluid (CSF) of children with meningitis. STUDY DESIGN. Prospective descriptive study involving children undergoing lumbar puncture during evaluation for meningitis. METHODS: CSF concentrations of defensins and lactoferrin were determined using enzyme-linked immunosorbent assays on 19 children with bacterial meningitis, 31 children with aseptic meningitis, and 32 control children found to have normal CSF during evaluation for meningitis. Pertinent clinical and laboratory data were gathered on all children. RESULTS: CSF concentrations of both defensins and lactoferrin were elevated markedly in children with bacterial and aseptic meningitis, compared with control children. No control subject had detectable levels of defensins in the CSF. Lactoferrin was undetectable in the CSF of 31 of 32 control subjects. Defensin and lactoferrin levels were significantly higher in the CSF of children with bacterial meningitis than in those with aseptic meningitis. Defensin levels in the CSF of children with bacterial meningitis ranged from 128 ng/mL to 99 430 ng/mL with a mean of 30 311 ng/mL (SD +/- 28 865) and a median of 23 042 ng/mL. Defensin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 1675 ng/mL with a mean of 227 ng/mL (SD +/- 433) and a median of 23 ng/mL. A significant correlation was found between defensin levels in the CSF and the total leukocyte count and the absolute neutrophil count in the CSF of children with bacterial meningitis. Lactoferrin levels in the CSF of children with bacterial meningitis ranged from 184 ng/mL to 31 412 ng/mL with a mean of 13 209 ng/mL (SD +/- 9644) and a median of 10 382 ng/mL. Lactoferrin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 2715 ng/mL with a mean of 1042 ng/mL (SD +/- 878) and a median of 852 ng/mL. No correlation was found between lactoferrin level in the CSF and the total leukocyte count or the absolute neutrophil count in the CSF of children with bacterial meningitis. In our study population, the sum total of CSF defensins and lactoferrin was found to be highly sensitive and specific in delineating bacterial from aseptic meningitis when compared with standard CSF studies. CONCLUSIONS: Significant elevations of defensins and lactoferrin, indicative of endogenous local antimicrobial peptide and polypeptide release, are found in the CSF of children with meningitis. We speculate that elevations in these antimicrobial molecules may reflect the intensity of the host response. Defensins seem to parallel neutrophil activation more closely than lactoferrin. Cumulative levels of CSF defensins and lactoferrin clearly distinguished bacterial meningitis from aseptic meningitis and control patients. Further investigation is warranted to determine the usefulness of measuring defensins and lactoferrin as a diagnostic tool and therapeutic monitor in the evaluation of children with meningitis.

[Immunoenzyme method of lactoferrin analysis]. / Immunofermentnyi metod opredeleniia laktoferrina.

The authors propose a highly effective method for isolation of lactoferrin from female colostrum. Lactoferrin is measured by the sandwich enzyme immunoassay. Its concentrations in the serum, urine, amniotic and cerebrospinal fluid in health are measured. The advantages of the method for lactoferrin purification and the practical significance of this protein are discussed.

Age-related alterations of the blood-brain-barrier (bbb) permeability to protein molecules of different size.

Transfer processes of differently sized protein molecules across the blood-brain-barrier (bbb) were studied in "normal" , respectively, diseased elderly humans. Lumbar CSF/serum ratio of albumin contents increased significantly with age in a control group, but it was diminished in patients suffering from inflammatory or non-inflammatory diseases. Ratio of alpha 2-macroglobulin contents increased with age, especially in the diseased elderly. Concentration ratios of lysozyme, orosomucoid, lactoferrin, IgG, IgA and IgM decreased with age in a control group and especially decreased in a patient group. The data are discussed with respect to two different transfer processes which appeared to be altered in age and/or disease processes.

Beta 2-microglobulin, lysozyme and lactoferrin in cerebrospinal fluid in patients with lymphoma or leukaemia: relationship to CNS involvement and the effect of prophylactic intrathecal treatment with methotrexate.

Central nervous system (CNS) involvement in patients with leukaemia or lymphoma presents a diagnostic problem. This study was conducted to test whether combined measurements of various cellular markers such as beta 2-microglobulin (beta 2m), lactoferrin (LF) and lysozyme (LYS) in the cerebrospinal fluid (CSF) might aid in the diagnosis of CNS involvement in such patients. Forty-two patients were studied. Sixteen were considered to have CNS involvement and 26 showed no signs of such involvement. In the group with symptoms or signs of CNS involvement, nine patients out of 12 had increased total protein in CSF, 14 of 14 increased beta 2m, 14 of 16 increased LYS and five of 15 increased LF. In patients without CNS involvement total protein was increased in four of 25, beta 2m in three of 21, LYS in four of 28 and LF in one of 28 patients. The differences were statistically significant (P less than 0.01, P less than 0.001, P less than 0.001 and P less than 0.05, respectively). Prophylactic intrathecal methotrexate treatment in patients with acute lymphoblastic leukaemia caused an increase in the CSF of beta 2m, LYS and LF but not of total protein, which may reflect a drug-induced inflammatory reaction in the CNS. We conclude that combined measurements of the three cell markers add to our understanding of the cellular reaction to malignant cells in the CNS in leukaemia and lymphoma and may be valuable supplements in the diagnosis of this CNS involvement.

Lactoferrin, C-reactive protein, alpha-1-antitrypsin and immunoglobulin GA in cerebrospinal fluid in meningitis.

Cerebrospinal fluid measurements of lactoferrin and alpha-1-antitrypsin showed significant elevation in bacterial meningitis in children. 8 of 10 lactoferrin values and 6 of 11 alpha-1-antitrypsin values were above the upper range of controls. Both proteins correlated well with the total number of leukocytes in the cerebrospinal fluid. C-reactive protein, measured by either agglutination or radial immunodiffusion in the cerebrospinal fluid, failed to demonstrate any usefulness in diagnosing bacterial meningitis. Neither elevated serum C-reactive protein in cases of bacterial meningitis, nor sepsis, gave detectable concentrations of C-reactive protein in the cerebrospinal fluid.

Eosinophil cationic protein (ECP) in the cerebrospinal fluid.

ECP (eosinophil cationic protein) has been measured by means of a specific radioimmunoassay in the cerebrospinal fluid (CSF) from 210 individuals with various diseases affecting the central nervous system. In the same specimens lactoferrin and albumin were measured as well, as indicators of neutrophil-involved inflammation and damage to the blood-brain barrier. From a patient reference group (n = 39) the upper "normal" limit for ECP was estimated to 1.7 microgram/l. In patients with acute cerebrovascular disease (n = 108) ECP levels were elevated in 38% of the cases which was a significantly (P less than 0.001) greater proportion than seen for lactoferrin (7%). In patients with acute infections of the CNS (n = 30) 67% had raised ECP levels with significantly higher levels (P less than 0.001) in those having bacterial infections. The ECP levels were significantly correlated (P less than 0.001) to the lactoferrin-levels in the whole infectious group. In patients with tumours (n = 25) raised levels of ECP were found in 67% of those with malignant and in 6% of those having benign tumours. This difference was statistically significant (P = 0.001). The ECP levels were closely related to those of lactoferrin (P less than 0.001) and albumin (P less than 0.005). Of the patients with multiple sclerosis (n = 19) 25% had raised ECP levels. This proportion was not significantly different from those having raised lactoferrin levels. In three patients extremely high ECP levels (70-455 micrograms/l) were found and a causal relationship between ECP and the brain tissue damage in these patients is suggested. In comparison with the neutrophil-related data the findings suggest a preferential involvement of eosinophils in some diseases affecting the central nervous system.

Lactoferrin, lysozyme, and beta 2-microglobulin levels in cerebrospinal fluid: differential indices of CNS inflammation.

The CSF levels of lactoferrin, lysozyme, and beta 2-microglobulin (beta 2 mu) were measured in patients with evident, probable, or possible inflammatory CNS reactions and compared to those found in neurologically apparently healthy patients. Patients with viral CNS infections had significantly raised beta 2 mu and lysozyme levels but normal lactoferrin levels, indicating a local activation of lymphocytes and monocytes but not of granulocytes. Patients with bacterial CNS infections had significantly raised levels of all three cell markers, but the increase of lysozyme and lactoferrin was relatively more pronounced than that of beta 2 mu, indicating that the inflammatory response to bacterial agents is dominated by monocytes and granulocytes. Patients with primary or secondary malignant brain tumors were characterized by a moderate increase of beta 2 mu and a considerable increase in both lysozyme and lactoferrin, i.e., the same protein pattern as observed in bacterial CNS infection. The lysozyme levels were moderately increased in half the patients with benign cerebral tumors while the levels of beta 2 mu and lactoferrin were normal, indicating that benign and malignant brain tumors induce different local inflammatory CNS reactions. Half the patients with pituitary gland adenoma had elevated beta 2 mu and lysozyme levels but normal lactoferrin levels, suggesting that immunological mechanisms are associated with the adenoma development. Patients with MS had moderately but significantly raised CSF levels of beta 2 mu and lysozyme and a third of them also had raised levels of lactoferrin, a protein pattern suggesting a low-active inflammatory process in CNS involving mononuclears and granulocytes. A similar protein pattern was found in Guillain-Barré syndrome. In cerebrosarcoidosis we noted considerably increased lysozyme and beta 2 mu but normal lactoferrin levels, consistent with the idea that the sarcoid granuloma mass is dominated by monocytic inflammatory cells. The data obtained indicate a clinical value of lactoferrin, lysozyme, and beta 2 mu as differential indices of inflammatory cell reactions taking place in various CNS processes.

Lactoferrin, lysozyme, and beta 2-microglobulin in cerebrospinal fluid. Elevated levels in patients with acute cerebrovascular lesions as indices of inflammation.

Serial determinations of beta 2-microglobulin, lactoferrin and lysozyme in CSF were performed in 14 patients with acute cerebrovascular lesions. Marked elevations were noted in patients with cerebral bleeding or hemorrhagic infarction. Patients with infarction without signs of bleeding or with cerebrovascular lesions undetectable by computed tomography also had an increase in these proteins. The increases in CSF of beta 2-microglobulin, lactoferrin and lysozyme could not be explained by a damaged blood-brain barrier but was believed to be a local product of the central nervous system. Peak levels of lactoferrin and lysozyme were noted on day 2-3 after onset of symptoms. Lactoferrin then declined while lysozyme remained elevated for another few days. beta 2-microglobulin gradually increased reaching peak levels on day 4-5 and remained elevated even 2 weeks after the onset of symptoms. We suggest that the increases of lactoferrin, lysozyme and beta 2-microglobulin reflect various inflammatory reactions mediated by granulocytes, macrophages and lymphocytes, respectively.